Deep mutational scanning of influenza A virus NEP reveals pleiotropic mutations in its N-terminal domain.

The influenza A virus nuclear export protein (NEP) is a multifunctional protein that is essential for the viral life cycle and has very high sequence conservation. However, since the open reading frame of NEP largely overlaps with that of another influenza viral protein, non-structural protein 1, it is difficult to infer the functional constraints of NEP based on sequence conservation analysis. Besides, the N-terminal of NEP is structurally disordered, which further complicates the understanding of its function. Here, we systematically measured the replication fitness effects of >1,800 mutations of NEP. Our results show that the N-terminal domain has high mutational tolerance. Additional experiments demonstrate that N-terminal domain mutations pleiotropically affect viral transcription and replication dynamics, host cellular responses, and mammalian adaptation of avian influenza virus. Overall, our study not only advances the functional understanding of NEP, but also provides insights into its evolutionary constraints.

Conditional convolutional GAN-based adaptive demodulator for OAM-SK-FSO communication.

The perturbation of atmosphere turbulence is a significant challenge in orbital angular momentum shift keying-based free space optical communication (OAM-SK-FSO). In this study, we propose an adaptive optical demodulation system based on deep learning techniques. A conditional convolutional GAN (ccGAN) network is applied to recover the distorted intensity pattern and assign it to its specified class. Compared to existing methods based on convolutional neural networks (CNNs), our network demonstrates powerful capability in recovering the distorted light beam, resulting in a higher recognition accuracy rate under the same conditions. The average recognition accuracy rates are 0.9928, 0.9795 and 0.9490 when the atmospheric refractive index structure constant $C_n^2$ is set at 3 × 10-13, 4.45 × 10-13, 6 × 10-13m-2/3, respectively. The ccGAN network provides a promising potential tool for free space optical communication.

Using a longitudinal network structure to subgroup depressive symptoms among adolescents.

BACKGROUND: Network modeling has been proposed as an effective approach to examine complex associations among antecedents, mediators and symptoms. This study aimed to investigate whether the severity of depressive symptoms affects the multivariate relationships among symptoms and mediating factors over a 2-year longitudinal follow-up. METHODS: We recruited a school-based cohort of 1480 primary and secondary school students over four semesters from January 2020 to December 2021. The participants (n = 1145) were assessed at four time points (ages 10-13 years old at baseline). Based on a cut-off score of 5 on the 9-item Patient Health Questionnaire at each time point, the participants were categorized into the non-depressive symptom (NDS) and depressive symptom (DS) groups. We conducted network analysis to investigate the symptom-to-symptom influences in these two groups over time. RESULTS: The global network metrics did not differ statistically between the NDS and DS groups at four time points. However, network connection strength varied with symptom severity. The edge weights between learning anxiety and social anxiety were prominently in the NDS group over time. The central factors for NDS and DS were oversensitivity and impulsivity (3 out of 4 time points), respectively. Moreover, both node strength and closeness were stable over time in both groups. CONCLUSIONS: Our study suggests that interrelationships among symptoms and contributing factors are generally stable in adolescents, but a higher severity of depressive symptoms may lead to increased stability in these relationships.

Evolution of the gene regulatory network of body axis by enhancer hijacking in amphioxus.

A central goal of evolutionary developmental biology is to decipher the evolutionary pattern of gene regulatory networks (GRNs) that control embryonic development, and the mechanism underlying GRNs evolution. The Nodal signaling that governs the body axes of deuterostomes exhibits a conserved GRN orchestrated principally by Nodal, Gdf1/3, and Lefty. Here we show that this GRN has been rewired in cephalochordate amphioxus. We found that while the amphioxus Gdf1/3 ortholog exhibited nearly no embryonic expression, its duplicate Gdf1/3-like, linked to Lefty, was zygotically expressed in a similar pattern as Lefty. Consistent with this, while Gdf1/3-like mutants showed defects in axial development, Gdf1/3 mutants did not. Further transgenic analyses showed that the intergenic region between Gdf1/3-like and Lefty could drive reporter gene expression as that of the two genes. These results indicated that Gdf1/3-like has taken over the axial development role of Gdf1/3 in amphioxus, possibly through hijacking Lefty enhancers. We finally demonstrated that, to compensate for the loss of maternal Gdf1/3 expression, Nodal has become an indispensable maternal factor in amphioxus and its maternal mutants caused axial defects as Gdf1/3-like mutants. We therefore demonstrated a case that the evolution of GRNs could be triggered by enhancer hijacking events. This pivotal event has allowed the emergence of a new GRN in extant amphioxus, presumably through a stepwise process. In addition, the co-expression of Gdf1/3-like and Lefty achieved by a shared regulatory region may have provided robustness during body axis formation, which provides a selection-based hypothesis for the phenomena called developmental system drift.

Pros and Cons in Various Immobilization Techniques and Carriers for Enzymes.

In recent years, enzyme immobilization technology has been developed, and studies on immobilized enzyme materials have become very prominent. With the immobilization technique, enzymes and compatible carrier materials are combined or enzyme crystals/aggregates are used in a carrier-free fashion, by physical, chemical, or biochemical methods. As a kind of biocatalyst, immobilized enzymes can catalyze certain chemical reactions with high selectivity and high efficiency under relatively mild reaction conditions and eliminate pollution to the environment. Considering the current status and applications of immobilized enzyme technology and materials emerging in the last 5 years, this mini-review introduces the advantages and disadvantages of various enzyme immobilization techniques with carriers as well as the pros and cons of different materials for immobilization. The future prospects of immobilization technology and carrier materials are outlined, aiming to provide a reference for further research and applications of sustainable technology.

Factual and counterfactual learning in major adolescent depressive disorder, evidence from an instrumental learning study.

BACKGROUND: The incidence of adolescent depressive disorder is globally skyrocketing in recent decades, albeit the causes and the decision deficits depression incurs has yet to be well-examined. With an instrumental learning task, the aim of the current study is to investigate the extent to which learning behavior deviates from that observed in healthy adolescent controls and track the underlying mechanistic channel for such a deviation. METHODS: We recruited a group of adolescents with major depression and age-matched healthy control subjects to carry out the learning task with either gain or loss outcome and applied a reinforcement learning model that dissociates valence (positive v. negative) of reward prediction error and selection (chosen v. unchosen). RESULTS: The results demonstrated that adolescent depressive patients performed significantly less well than the control group. Learning rates suggested that the optimistic bias that overall characterizes healthy adolescent subjects was absent for the depressive adolescent patients. Moreover, depressed adolescents exhibited an increased pessimistic bias for the counterfactual outcome. Lastly, individual difference analysis suggested that these observed biases, which significantly deviated from that observed in normal controls, were linked with the severity of depressive symoptoms as measured by HAMD scores. CONCLUSIONS: By leveraging an incentivized instrumental learning task with computational modeling within a reinforcement learning framework, the current study reveals a mechanistic decision-making deficit in adolescent depressive disorder. These findings, which have implications for the identification of behavioral markers in depression, could support the clinical evaluation, including both diagnosis and prognosis of this disorder.

Amphioxus Gli knockout disrupts the development of left-right asymmetry but has limited impact on neural patterning.

The Gli transcription factors are the primary mediators of Hedgehog (Hh) signaling. Vertebrate genomes contain multiple Gli paralogues with different functions downstream of Hh signal receipt, in part explaining the complexity of cellular responses to Hh that allow concentration-dependent target gene activation. Amphioxus is a chordate that split from the vertebrate lineage early in the evolution of chordates, before the genome duplications that occurred in early vertebrate evolution. It has a single Gli gene whose transcripts can be alternately spliced to yield two protein isoforms called GliS and GliL. We generated two knockout mutations in amphioxus Gli, one that affects the whole gene and a second that only affects GliL. Both knockouts showed major morphological and molecular defects in the development of left-right asymmetry, a phenotype that is similar but not identical to that previously found in Hh mutants. Hh signaling also patterns the amphioxus neural tube. Here, however, knockout of GliL showed no identifiable phenotype, while knockout of the full gene showed only small changes to the expression of one gene family, Olig. Other genes that were prominently affected by Hh knockout were not altered in expression in either knockout. Reasons for the differences between Hh and Gli knockouts in the pharynx and neural tube are discussed in the context of the likely different functions of amphioxus Gli isoforms. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00195-w.

Leveraging vaccination-induced protective antibodies to define conserved epitopes on influenza N2 neuraminidase.

There is growing appreciation for neuraminidase (NA) as an influenza vaccine target; however, its antigenicity remains poorly characterized. In this study, we isolated three broadly reactive N2 antibodies from the plasmablasts of a single vaccinee, including one that cross-reacts with NAs from seasonal H3N2 strains spanning five decades. Although these three antibodies have diverse germline usages, they recognize similar epitopes that are distant from the NA active site and instead involve the highly conserved underside of NA head domain. We also showed that all three antibodies confer prophylactic and therapeutic protection in vivo, due to both Fc effector functions and NA inhibition through steric hindrance. Additionally, the contribution of Fc effector functions to protection in vivo inversely correlates with viral growth inhibition activity in vitro. Overall, our findings advance the understanding of NA antibody response and provide important insights into the development of a broadly protective influenza vaccine.

Stringent and complex sequence constraints of an IGHV1-69 broadly neutralizing antibody to influenza HA stem.

IGHV1-69 is frequently utilized by broadly neutralizing influenza antibodies to the hemagglutinin (HA) stem. These IGHV1-69 HA stem antibodies have diverse complementarity-determining region (CDR) H3 sequences. Besides, their light chains have minimal to no contact with the epitope. Consequently, sequence determinants that confer IGHV1-69 antibodies with HA stem specificity remain largely elusive. Using high-throughput experiments, this study reveals the importance of light-chain sequence for the IGHV1-69 HA stem antibody CR9114, which is the broadest influenza antibody known to date. Moreover, we demonstrate that the CDR H3 sequences from many other IGHV1-69 antibodies, including those to the HA stem, are incompatible with CR9114. Along with mutagenesis and structural analysis, our results indicate that light-chain and CDR H3 sequences coordinately determine the HA stem specificity of IGHV1-69 antibodies. Overall, this work provides molecular insights into broadly neutralizing antibody responses to influenza virus, which have important implications for universal influenza vaccine development.

TransIntegrator: capture nearly full protein-coding transcript variants via integrating Illumina and PacBio transcriptomes.

Genes have the ability to produce transcript variants that perform specific cellular functions. However, accurately detecting all transcript variants remains a long-standing challenge, especially when working with poorly annotated genomes or without a known genome. To address this issue, we have developed a new computational method, TransIntegrator, which enables transcriptome-wide detection of novel transcript variants. For this, we determined 10 Illumina sequencing transcriptomes and a PacBio full-length transcriptome for consecutive embryo development stages of amphioxus, a species of great evolutionary importance. Based on the transcriptomes, we employed TransIntegrator to create a comprehensive transcript variant library, namely iTranscriptome. The resulting iTrancriptome contained 91 915 distinct transcript variants, with an average of 2.4 variants per gene. This substantially improved current amphioxus genome annotation by expanding the number of genes from 21 954 to 38 777. Further analysis manifested that the gene expansion was largely ascribed to integration of multiple Illumina datasets instead of involving the PacBio data. Moreover, we demonstrated an example application of TransIntegrator, via generating iTrancriptome, in aiding accurate transcriptome assembly, which significantly outperformed other hybrid methods such as IDP-denovo and Trinity. For user convenience, we have deposited the source codes of TransIntegrator on GitHub as well as a conda package in Anaconda. In summary, this study proposes an affordable but efficient method for reliable transcriptomic research in most species.

Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

Betacoronaviruses encode an internal (I) gene via an alternative reading frame within the nucleocapsid gene, called ORF8b for Middle-East respiratory syndrome coronavirus (MERS-CoV) and ORF9b for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that proteins 8b and 9b are involved in evading multiple innate immune signaling pathways. However, their roles in mediating pathogenesis in infected animals have not been determined. In this study, we abrogated the expression of protein 8b in MERS-CoV and protein 9b in SARS-CoV-2. Using mouse models of MERS-CoV and SARS-CoV-2 infection, we found that MERS-CoV lacking protein 8b expression was more virulent, while SARS-CoV-2 lacking protein 9b expression was attenuated compared with the respective wild-type viruses. Upon further analysis, we detected increased levels of type I interferon and enhanced infiltration of immune cells to the lungs of mice infected with MERS-CoV lacking protein 8b expression. These data suggest that the I protein of MERS-CoV plays a role in limiting pathogenesis while that of SARS-CoV-2 enhances disease severity. IMPORTANCE The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.

An explainable language model for antibody specificity prediction using curated influenza hemagglutinin antibodies.

Despite decades of antibody research, it remains challenging to predict the specificity of an antibody solely based on its sequence. Two major obstacles are the lack of appropriate models and inaccessibility of datasets for model training. In this study, we curated a dataset of >5,000 influenza hemagglutinin (HA) antibodies by mining research publications and patents, which revealed many distinct sequence features between antibodies to HA head and stem domains. We then leveraged this dataset to develop a lightweight memory B cell language model (mBLM) for sequence-based antibody specificity prediction. Model explainability analysis showed that mBLM captured key sequence motifs of HA stem antibodies. Additionally, by applying mBLM to HA antibodies with unknown epitopes, we discovered and experimentally validated many HA stem antibodies. Overall, this study not only advances our molecular understanding of antibody response to influenza virus, but also provides an invaluable resource for applying deep learning to antibody research.

Stringent and complex sequence constraints of an IGHV1-69 broadly neutralizing antibody to influenza HA stem.

IGHV1-69 is frequently utilized by broadly neutralizing influenza antibodies to the hemagglutinin (HA) stem. These IGHV1-69 HA stem antibodies have diverse complementarity-determining region (CDR) H3 sequences. Besides, their light chains have minimal to no contact with the epitope. Consequently, sequence determinants that confer IGHV1-69 antibodies with HA stem specificity remain largely elusive. Using high-throughput experiments, this study revealed the importance of light chain sequence for the IGHV1-69 HA stem antibody CR9114, which is the broadest influenza antibody known to date. Moreover, we demonstrated that the CDR H3 sequences from many other IGHV1-69 antibodies, including those to HA stem, were incompatible with CR9114. Along with mutagenesis and structural analysis, our results indicate that light chain and CDR H3 sequences coordinately determine the HA stem specificity of IGHV1-69 antibodies. Overall, this work provides molecular insights into broadly neutralizing antibody responses to influenza virus, which have important implications for universal influenza vaccine development.

Occipital bone defect caused by neurofibromatosis type I: A case report.

RATIONALE: The synergistic effect between nonmalignant lesions can also cause a serious impact on patient survival. This disease involves different organ systems and presents with a variety of clinical manifestations, such as schwannoma, depigmentation, low-grade glioma, and skeletal abnormalities. We report a case of type I neurofibromatosis with an occipital bone defect. PATIENT CONCERNS: We report a case of a 50-year-old man with type I neurofibromatosis with occipital bone defect. DIAGNOSIS: The patient was accepted by the local hospital due to sudden right upper limb weakness accompanied by jitter without recognizable cause or inducement. A computerized tomography scan at a local hospital suggested subcutaneous neurofibromatosis with a left occipital cranial defect with thinning bone. On admission physical examination, diffuse multiple masses could be seen throughout the body and head of different sizes and composed of soft and tough textures. The largest one was located in the posterior occipital bone, approximately 8*8 cm in size, with a child tumor and tough texture. Multiple café-au-lait spots could be found on the chest and back, and multiple freckles can be seen in the armpit. The patient underwent surgery. Postoperative pathology showed a spindle cell tumor, which was determined to be neurofibromatosis type I according to immunopathology and clinical data. INTERVENTIONS: The patient was admitted for surgical treatment. During the operation, the scalp mass was completely abducted and the tumor tissue at the skull defect was sharply separated. Postoperative pathology showed that the peripheral margin and the bottom margin were cleaned. OUTCOMES: Computerized tomography and magnetic resonance imaging showed that the tumor was completely. There were not any surgical complications. The patient recovered well, was cured and was dismissed from the hospital. LESSONS: The synergistic effect between nonmalignant lesions can also cause a serious impact on patient survival to encourage early medical intervention. The clinical presentation of neurofibromatosis type I am usually nonmalignant, and in this case, involvement of the skull with bone defect is very rare. Therefore, it is necessary to accumulate relevant cases, reveal the pathogenesis of the disease, predict the development and outcome, and provide more evidence for early therapeutic intervention of similar patients in the future.

Soil water depletion patterns in rainfed apple orchards and wheat fields.

Agricultural production in the Weibei rainfed highland, Northwest China, is challenged by severe drought and water shortages. While the land use pattern has shifted gradually from crop production to orchard farming in Weibei, little is known about the influence of fruit industry development on regional water resources and the rationality of planting orchards. Here, we characterized soil water depletion patterns in rainfed orchards and farmlands to evaluate the occurrence of soil desiccation under land use conversion from farmlands to orchards in Weibei. Soil moisture dynamics were monitored in the 0-150 cm soil profiles of different aged Red Fuji apple orchards (young: 7 years, mature: 13 years, old: 22 years) and long-term cultivated winter wheat fields. We measured soil moisture content by oven-drying method in the middle of each month during the growing season of apple trees (March-September 2019). The over-depletion and depletion of soil water were analyzed to evaluate water stress and differential water depletion by distinct vegetation, respectively. The soil desiccation index was used to determine the occurrence of dry soil layers. Water stress was only observed at the 0-70-cm soil depths in the old orchards (mid-June) and farmlands (mid-May-mid-July). Water depletion took place at deeper depths for longer periods in the older orchards than in the younger orchards. Soil desiccation was absent in the young orchards, with mild desiccation at the 0-80-cm soil depths in the mature and old orchards in mid-June. The desiccation intensity was mild at the 0-60-cm soil depths in mid-April-mid-May, intense at the 0-150-cm soil depths in mid-June, and moderate at the 20-150-cm soil depths in mid-July. Results of this study demonstrate the mitigation of water stress and soil desiccation following conversion from wheat fields to apple orchards, which verifies the rationality of planting orchards in the rainfed highland area. Our findings provide strong support for developing a novel model of agro-industrial development, ecological construction, and sustainable economy in the vast arid and semi-arid areas of Northwest China.

Mutational fitness landscape of human influenza H3N2 neuraminidase.

Influenza neuraminidase (NA) has received increasing attention as an effective vaccine target. However, its mutational tolerance is not well characterized. Here, the fitness effects of >6,000 mutations in human H3N2 NA are probed using deep mutational scanning. Our result shows that while its antigenic regions have high mutational tolerance, there are solvent-exposed regions with low mutational tolerance. We also find that protein stability is a major determinant of NA mutational fitness. The deep mutational scanning result correlates well with mutational fitness inferred from natural sequences using a protein language model, substantiating the relevance of our findings to the natural evolution of circulating strains. Additional analysis further suggests that human H3N2 NA is far from running out of mutations despite already evolving for >50 years. Overall, this study advances our understanding of the evolutionary potential of NA and the underlying biophysical constraints, which in turn provide insights into NA-based vaccine design.

Exploring structural and functional alterations in drug-naïve obsessive-compulsive disorder patients: An ultrahigh field multimodal MRI study.

BACKGROUND: Brain structural and functional alterations have been reported in obsessive-compulsive disorder (OCD) patients; however, these findings were inconsistent across studies due to several limitations, including small sample sizes, different inclusion/exclusion criteria, varied demographic characteristics and symptom dimensions, comorbidity, and medication status. Prominent and replicable neuroimaging biomarkers remain to be discovered. METHODS: This study explored the gray matter structure, neural activity, and white matter microstructure differences in 40 drug-naïve OCD patients and 57 matched healthy controls using ultrahigh field 7.0 T multimodal magnetic resonance imaging, which increased the spatial resolution and detection power. We also evaluated correlations among different modalities, imaging features and clinical symptoms. RESULTS: Drug-naïve OCD patients exhibited significantly increased gray matter volume in the frontal cortex, especially in the orbitofrontal cortex, as well as volumetric reduction in the temporal lobe, occipital lobe and cerebellum. Increased neural activities were observed in the cingulate gyri and precuneus. Increased temporal-middle cingulate and posterior cingulate-precuneus functional connectivities and decreased frontal-middle cingulate connectivity were further detected. Decreased fractional anisotropy values were found in the cingulum-hippocampus gyrus and inferior fronto-occipital fascicle in OCD patients. Moreover, significantly altered imaging features were related to OCD symptom severity. Altered functional and structural neural connectivity might influence compulsive and obsessive features, respectively. CONCLUSIONS: Altered structure and function of the classical cortico-striato-thalamo-cortical circuit, limbic system, default mode network, visual, language and sensorimotor networks play important roles in the neurophysiology of OCD.

Molecular analysis of a public cross-neutralizing antibody response to SARS-CoV-2.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concerns (VOCs) continue to emerge, cross-neutralizing antibody responses become key toward next-generation design of a more universal COVID-19 vaccine. By analyzing published data from the literature, we report here that the combination of germline genes IGHV2-5/IGLV2-14 represents a public antibody response to the receptor-binding domain (RBD) that potently cross-neutralizes a broad range of VOCs, including Omicron and its sub-lineages. Detailed molecular analysis shows that the complementarity-determining region H3 sequences of IGHV2-5/IGLV2-14-encoded RBD antibodies have a preferred length of 11 amino acids and a conserved HxIxxI motif. In addition, these antibodies have a strong allelic preference due to an allelic polymorphism at amino acid residue 54 of IGHV2-5, which is located at the paratope. These findings have important implications for understanding cross-neutralizing antibody responses to SARS-CoV-2 and its heterogenicity at the population level as well as the development of a universal COVID-19 vaccine.

Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase.

Neuraminidase (NA) of human influenza H3N2 virus has evolved rapidly and been accumulating mutations for more than half-century. However, biophysical constraints that govern the evolutionary trajectories of NA remain largely elusive. Here, we show that among 70 natural mutations that are present in the NA of a recent human H3N2 strain, >10% are deleterious for an ancestral strain. By mapping the permissive mutations using combinatorial mutagenesis and next-generation sequencing, an extensive epistatic network is revealed. Biophysical and structural analyses further demonstrate that certain epistatic interactions can be explained by non-additive stability effect, which in turn modulates membrane trafficking and enzymatic activity of NA. Additionally, our results suggest that other biophysical mechanisms also contribute to epistasis in NA evolution. Overall, these findings not only provide mechanistic insights into the evolution of human influenza NA and elucidate its sequence-structure-function relationship, but also have important implications for the development of next-generation influenza vaccines.